PROJECT SUMMARY Nucleosomes are the fundamental repeating units of chromatin, comprised of four core histone proteins that are subject to a variety of post-translational modifications (PTMs, e.g. lysine methylation and acetylation). Epigenetic reader proteins coordinate the cellular response to these PTMs by influencing chromatin compaction and gene expression through domains that recognize specific PTMs. A rapidly expanding area of research has shown that reader proteins often contain combinations of domains (i.e. multivalency) in order to simultaneously translate multiple PTMs that decorate a single nucleosome. Readout of this ?histone code? has profound functional outcomes for cellular functions (e.g. cell cycle regulation) as well as disease states (e.g. cancer and others). Despite increasing recognition that quantifying combinations of epigenetic marks can improve predictive validity in biomarker studies and also increase the magnitude of response to epigenetic inhibitor treatment, the dearth of available technologies to quantify co-occurring PTMs has impeded scientific discovery in this field. A major limitation for developing assays to quantify combinatorial PTMs has been the lack of control reagents that offer sufficient resolution to detect the combinations of PTMs that are read by multivalent readers. Readouts based on histone proteins (mass spectrometry, histone ELISAs) require elaborate sample processing protocols and simply cannot detect epigenetic marks in trans (e.g. PTMs on different histone tails within a nucleosome, histone-DNA interactions, etc.). Approaches capable of assaying PTMs at nucleosomal resolution are low throughput, laborious, and/or not quantitative (e.g. single nucleosome imaging, re-ChIP). Clearly, development of streamlined, high-throughput, quantitative assays using defined nucleosomal calibrants is urgently needed to decipher combinatorial histone codes and accelerate epigenetic therapies. Here, we propose to develop ?QuantiNucTM?, a high-throughput platform of nucleosome-calibrated assays to measure PTMs from biological samples with high precision. The innovation of this proposal is the novel application of modified recombinant nucleosome calibrants to enable minimal sample processing and reliable quantification of both single and combinatorial PTMs. In Aim 1, we will develop a combinatorially-modified nucleosome and employ it to optimize ELISAs (low-throughput but widely accessible applications) as well as AlphaLISAs (bead-based proximity assays for high-throughput applications). The optimized reagents and assays developed in Aim 1 will be used for proof-of-principle biological validation in Aim 2, where we will apply QuantiNuc to recapitulate the unique biology of combinatorial PTMs. Completion of these aims will demonstrate feasibility for developing an expanded QuantiNuc platform in Phase II to commercialize assays that will help decipher the histone code, enable biomarker discovery, and facilitate drug development.